The terpene indole alkaloids are a diverse group of molecules with a range of chemical structures and medicinal uses. Understanding the enzymes that catalyze natural product biosynthesis may enable production in more tractable host organisms, and may also allow reprogramming of biosynthetic pathways to produce "unnatural" natural products with improved pharmacological activities. Our laboratory seeks to understand, and ultimately harness, the metabolic pathways that direct the biosynthesis of plant-derived terpene indole alkaloids. This proposal describes the use of the terpene indole alkaloid biosynthetic pathway to make novel alkaloid structures. Specific Aim 1. The First Committed Step of Terpene Indole Alkaloid Biosynthesis This aim focuses on strictosidine synthase, the enzyme that catalyzes the first committed step of the terpene indole alkaloid biosynthetic pathway. The major focus of this aim is to understand and modify the substrate specificity of strictosidine synthase using rational and random mutagenesis. Synthesis of substrates, development of assays and design of mutants are described. Aim 1A Tryptamine Substrate Specificity Aim 1B Secologanin Substrate Specificity Aim 1C Altering Strictosidine Synthase Substrate Specificity Specific Aim 2. Later Steps in the Terpene Indole Alkaloid Pathway The substrate specificity of the second committed step of terpene indole biosynthesis, catalyzed by strictosidine glucosidase, will be examined. Precursor directed biosynthesis in C. roseus cell culture and plants are used to probe the substrate specificity of the later steps of the pathway. Alkaloid analogues are isolated and structurally characterized. Use of a substrate analogue to purify a later enzyme in the pathway is described. Aim 2A Substrate Specificity of Strictosidine Glucosidase Aim 2B Probing the Biosynthetic Pathway Specificity in vivo